title: Optically modulatable blue fluorescent proteins.
abstract: Blue fluorescent proteins (BFPs) offer visualization of protein location and behavior, but often suffer from high autofluorescent background and poor signal discrimination. Through dual-laser excitation of bright and photoinduced dark states, mutations to the residues surrounding the BFP chromophore enable long-wavelength optical modulation of BFP emission. Such dark state engineering enables violet-excited blue emission to be increased upon lower energy, green coillumination. Turning this green coillumination on and off at a specific frequency dynamically modulates collected blue fluorescence without generating additional background. Interpreted as transient photoconversion between neutral cis and anionic trans chromophoric forms, mutations tune photoisomerization and ground state tautomerizations to enable long-wavelength depopulation of the millisecond-lived, spectrally shifted dark states. Single mutations to the tyrosine-based blue fluorescent protein T203V/S205V exhibit enhanced modulation depth and varied frequency. Importantly, analogous single point mutations in the nonmodulatable BFP, mKalama1, creates a modulatable variant. Building modulatable BFPs offers opportunities for improved BFP signal discrimination vs background, greatly enhancing their utility.
authors.affiliation: School of Chemistry and Biochemistry, ‡School of Chemical and Biomolecular Engineering, and §Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , Atlanta, Georgia 30332-0400, United States.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Jablonski
authors.forename: Russell B
authors.initials: RB
authors.lastname: Vegh
authors.forename: Jung-Cheng
authors.initials: JC
authors.lastname: Hsiang
authors.forename: Bettina
authors.initials: B
authors.lastname: Bommarius
authors.forename: Yen-Cheng
authors.initials: YC
authors.lastname: Chen
authors.forename: Kyril M
authors.initials: KM
authors.lastname: Solntsev
authors.forename: Andreas S
authors.initials: AS
authors.lastname: Bommarius
authors.forename: Laren M
authors.initials: LM
authors.lastname: Tolbert
authors.forename: Robert M
authors.initials: RM
authors.lastname: Dickson
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D055620:Optical Phenomena
chemical_list: D008164:Luminescent Proteins